Locked aptamer hairpin integrated hybridization chain reaction for rapid, instrument-free biomarker detection

Abstract

In one aspect, the disclosure relates to a universal method for biomarker extraction and detection from clinical samples using magnetic nanoparticles (MNPs) coupled with a locked aptamer-initiator approach. The MNP bound locked aptamer undergoes a conformational change in the presence of the target biomarker and the unfolded initiator triggers a hybridization chain reaction with a structurally stabilized catalytic trimeric triplex DNAzyme hairpin. Amplicons from the hybridization chain reaction can be incubated with hemin to obtain a stable triplex structure that has horseradish peroxidase-like activity in the presence of H2O2 and a substrate molecule. The intensity of the signal is directly proportional to the amount of biomarker present. Binding one or more reagents on a nanoparticle can be coupled with this approach for use in a microfluidic or paper fluidic platform. In any of these aspects, the assay can detect as low as femtogram amounts of biomarker from the clinical sample.

What is claimed is: 
     
         1 . A method for detecting a biomarker in a clinical sample, the method comprising:
 (a) binding an aptamer locked in a first conformation to a magnetic nanoparticle (MNP);   (b) contacting the magnetic nanoparticle and aptamer with a clinical sample;   wherein presence of the biomarker in the clinical sample causes the aptamer to undergo a conformational change to a second conformation;   wherein the conformational change results the opening of an initiator which triggers a hybridization chain reaction with a trimeric triplex DNAzyme sequence incorporating a first hairpin and a second hairpin, wherein the first hairpin and the second hairpin are partially complementary to each other, forming a structurally stabilized catalytic trimeric triplex;   (c) contacting the structurally stabilized catalytic trimeric triplex with one or more reagents to produce a detectable signal; and   (d) detecting the signal.   
     
     
         2 . The method of  claim 1 , wherein the biomarker comprises a peptide, a protein, DNA, RNA, a carbohydrate, a lipid, a bacterium, a parasite, or a virus. 
     
     
         3 . The method of  claim 2 , wherein the biomarker comprises cardiac troponin I. 
     
     
         4 . The method of  claim 1 , further comprising contacting the magnetic nanoparticle, aptamer, and clinical sample with a third hairpin and a fourth hairpin. 
     
     
         5 . The method of  claim 4 , wherein performing the method forms a hyperbranched chain connected to the MNP, wherein the hyperbranched chain produces an increased signal intensity relative to an unbranched chain. 
     
     
         6 . The method of  claim 1 , wherein the magnetic nanoparticle comprises iron oxide. 
     
     
         7 . The method of  claim 6 , wherein the iron oxide is co-precipitated with silica and wherein the silica is amino functionalized using (3-aminopropyl)triethoxysilane (APTES). 
     
     
         8 . The method of  claim 7 , wherein the magnetic nanoparticle comprises a streptavidin coating, and wherein the streptavidin coating is conjugated to the APTES. 
     
     
         9 . The method of  claim 8 , wherein the aptamer is conjugated to biotin, and where the biotin binds to the streptavidin coating. 
     
     
         10 . The method of  claim 1 , wherein the one or more reagents comprise hemin, hydrogen peroxide, 3,3′,5,5′-tetramethylbenzidine (TMB), or any combination thereof, and a substrate, wherein the hemin induces stabilization of the catalytic trimeric triplex, and wherein the stabilized catalytic trimeric triplex has horseradish peroxidase activity in the presence of the hydrogen peroxide and the substrate. 
     
     
         11 . The method of  claim 1 , wherein the detectable signal comprises a fluorescence signal, a color, an electrochemical signal, a Raman signal, or another signal. 
     
     
         12 . The method of  claim 10 , wherein the substrate comprises 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and wherein the horseradish peroxidase activity produces a color in the presence of the hydrogen peroxide and the ABTS. 
     
     
         13 . The method of  claim 11 , wherein an intensity of the color is directly proportional to an amount of biomarker present in the clinical sample. 
     
     
         14 . The method of  claim 11 , wherein a change in intensity of the color is proportional to an amount of biomarker present in the clinical sample. 
     
     
         15 . The method of  claim 1 , wherein the one or more reagents comprise a gold particle, and wherein the detectable signal comprises fluorescent or Raman scattering, and wherein the gold particle comprises a gold nanostar or a gold nanosphere. 
     
     
         16 . The method of  claim 15 , wherein the gold particle comprises a silica shell and a plurality of dye molecules, wherein the dye molecules are encapsulated within the silica shell adjacent to a surface of the gold particle. 
     
     
         17 . The method of  claim 16 , wherein the dye molecules comprise NIR-780. 
     
     
         18 . The method of  claim 1 , wherein the method has a limit of detection (LoD) of about 10 femtomolar. 
     
     
         19 . A method for identifying myocardial infarction, the method comprising performing the method of  claim 1  on a clinical sample from a subject, wherein the biomarker is cardiac troponin I. 
     
     
         20 . The method of  claim 19 , wherein the myocardial infarction is asymptomatic.